No Federally sponsored research and development were used in making this invention.
Since they were first reported by Nuzzo and Allara in 1983, self-assembled monolayers (SAMs) composed of sulfur-terminated organic molecules adsorbed on and adherent to gold surfaces have shown broad utility in lubrication, electrochemistry, electronic and vibrational spectroscopy, photochemistry, diagnostics, the modification of biochemical membranes, catalysis, drug delivery, and facile modification of the absorptive properties of surfaces. (R. G. Nuzzo and D. L. Allara. Adsorption of bifunctional organic disulfides on gold surfaces. J. Am. Chem. Soc. 1983; 105: 4481-4483.) More recently, organic modifications of gold surfaces by SAMs have proven to be successful in nanotechnological biosensor applications, e.g., in commercially available chips for biomolecular interaction analysis with surface plasmon resonance. (S. Lxc3x6ffxc3xa5s, B. Johnsson, K. Tegendahl, and I. Rxc3x6nnberg. Colloids Surf. B 1993; 1: 83-89.) For example, Dijksma and coworkers have reported that an electrochemical immunosensor composed of self-assembled monolayers of cysteine or N-acetylcysteine on gold electrodes is useful for the detection of interferon-xcex3at the attomolar level. (M. Dijksma, B. Kamp, J. C. Hoogvliet, and W. P. van Bennekom. Development of an electrochemical immunosensor for direct detection of interferon-xcex3at the attomolar level. Analyt. Chem. 2001; 73: 901-907.) Similarly, Darder and coworkers have found that horseradish peroxidase retained its activity when immobilized onto a gold surface via a 3-thiopropionate tether and was useful as a peroxide biosensor. (M. Darder, K. Takeda, F. Pariente, E. Lorenzo, and H. D. Abruxc3x1a. Dithiobissuccinimidyl propionate as an anchor for assembling peroxidases at electrodes surfaces and its application in a H2O2 biosensor. Analyt. Chem. 1999; 71: 5530-5537.)
Likewise, poly- and oligo(ethylene glycols) (PEGs or OEGs, respectively; Structure 1, where R1 is MeO or HO and R2 is OH) have found widespread use in a variety of biotechnological and commercial applications, including the preparation of surfactants, ion-conducting materials, and conjugates of low and high molecular weight molecules. Investigators have found that these glycols provide good anchors for biological and non-biological receptor/reporter molecules or for ligands for biological and non-biological chelation or binding sites. Moreover, both PEGs and OEGs are known to reduce the nonspecific binding of proteins and other bioactive molecules to the surface to which they are conjugated. PEG and OEG derivatives are ideal for these applications because they are inexpensive, water soluble, stable, nonantigenic and non-immunogenic, and commercially available in a wide range of molecular weight distributions.
Structure 1: R1xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)xxe2x80x94CH2CH2xe2x80x94R2 
In addition, conjugation with more highly branched and dendritic poly- and oligo(ethylene glycols) has been reported to be useful for improving the stability of protein drugs. [(a) D.C. Tully and J. M. J. Frechet. Dendrimers at surfaces and interfaces: chemistry and applications. Chem. Commun. 2001; 1229-1239. (b) I. Fuke, T. Hayashi, Y. Tabata, and Y. Ikada. Synthesis of poly(ethylene glycol) derivatives with different branchings and their use for protein modification. J. Controlled Release 1994; 30: 27-34. (c) J. M. Harris, F. M. Veronese, P. Caliceti, and O. Schiavon, U.S. Pat. No. 5,932,462.]
The broad utility of both classes of reagents (i.e., SAMs and PEGs or OEGs) suggests that synergistic benefits would obtain if libraries of reagents were available that combined the beneficial attributes of a SAM with those a PEG or OEG and exhibited additional features, such as the presence of reactive or activated groups at one end of each PEG or OEG chain. This combination of attributes would enable attachment of one terminus of such a combined SAM-forming-OEG reagent to a metal surface, yielding a SAM-OEG reagent, and attachment of a biological or non-biological receptor, ligand or reporter moiety at each of the other activated or reactive termini of the combined SAM/OEG reagent. The literature reports that describe examples of combined SAM/OEG reagents are limited to disclosures of methods of synthesis of OEG conjugates of linear alkyl monothiols and the effects of structure on the stability and physico-chemical properties of the reagents and the SAMs formed from them. (S. Svedhem, C-A. Hollander, J. Shi, P. Konradsson, B. Liedberg, and S. C. T. Svensson. Synthesis of a series of oligo(ethylene glycol)-terminated alkanethiol amides designed to address structure and stability of biosensing surfaces. J. Org. Chem. 2001; 66: 4494-4503.) Thus, the known reagents are limited to alkyl monothiols that lack an activated or reactive terminus at the end of the OEG chain and other desirable attributes that would enhance their utility.
Clearly, significant biotechnological advances in a spectrum of areas would be possible if activated or reactive, oligo(ethylene glycol)-terminated reagents and OEG-terminated reagents conjugated with a biological or non-biological receptor, ligand or reporter moiety useful for preparing self-assembled monolayers on gold were available. The present invention addresses this need.
Moreover, significant therapeutic benefit would result if the pharmaceutical or pharmacological properties of a therapeutic agent were enhanced by conjugatively coupling with oligo(ethylene glycol)-terminated dithiolane reagents and OEG-terminated dithiolane reagents.
The invention is based upon the recognition that the availability of activated or reactive, oligo(ethylene glycol)-terminated dithiolane compositions suitable for use in preparing self-assembled monolayers on a metal would enable significant advances in the biotechnological arts.
Thus, the invention provides highly versatile tethers suitable for immobilization on a metal backbone, wherein one segment of the tether is a linear or branched oligo(ethylene glycol) residue and the other segment of the tether is an alkyl-substituted 1,2-dithiolane. Further, one terminus of each oligo(ethylene glycol) residue is activated or reactive, enabling the preparation of conjugates of the oligo(ethylene glycol)-terminated dithiolane compositions that are also suitable for immobilization on a metal backbone.
One embodiment of the present invention comprises linear or branched oligo(ethylene glycol)-terminated 3-alkyl-1,2-dithiolanes having the formula: 
wherein m is from about 3 to about 20; n is from 2 to about 6; OEG is shorthand for a linear oligoether having the general structure xe2x80x94(CH2CH2O)xxe2x80x94 wherein x is from 2 to about 100, or for a branched oligoether wherein each branch comprises a linear oligoether having this general structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide or hydrazide group; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological, ligand, sequestering, or reporter moiety. Examples of suitable reactive or activated substituents Z include an amino, guanidino, sulfhydryl, or activated ester moiety; a substituent that is reactive toward nucleophilic displacement, such as chloride, bromide, iodide, tosylate, tresylate, or mesylate; a group that is reactive toward nucleophilic addition, such as cyanate, isocyanate, thiocyanate, isothiocyanate, maleimide, oxirane, thiirane, or azirane; a carbonyl group; or a hydroxyl group.
A preferred embodiment comprises oligo(ethylene glycol)-terminated thioctic acid derivatives having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the general structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this general structure; one terminus of the OEG residue is covalently joined to the alkyl side chain of thioctic acid by a linker L, wherein L is amide or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety.
A particularly preferred embodiment comprises oligo(ethylene glycol)-terminated d-thioctic acid derivatives having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the alkyl side chain of d-thioctic acid by a linker L, wherein L is amide or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety.
Another embodiment of the present invention comprises oligo(ethylene glycol)-terminated 4-alkyl-1,2-dithiolanes having the formula: 
wherein m is from 3 to about 20; n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide, or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety. Examples of suitable reactive or activated substituents Z include an amino, guanidino, sulfhydryl, or activated ester moiety; a substituent that is reactive toward nucleophilic displacement, such as chloride, bromide, iodide, tosylate, tresylate, or mesylate; a group that is reactive toward nucleophilic addition, such as cyanate, isocyanate, thiocyanate, isothiocyante, maleimide, oxirane, thiirane, or azirane; a carbonyl group; or a hydroxyl group.
Also provided in accordance with the invention are conjugates of these activated polymers with a biological or non-biological receptor, ligand, sequestering, or reporter moiety such as a polypeptide, protein, enzyme, phospholipid, lipid, liposome, nucleoside, oligonucleotide, drug, dye, antibody reporter molecule, ligand, cyclodextrin, carceplex, boronate, biological membrane, or a surface of a solid material that is compatible with living organisms, tissue, or fluids. Further provided are methods for preparation of these conjugates.
Yet another particularly preferred embodiment comprises a conjugatively coupled oligomer composition comprising an oligo(ethylene glycol)-terminated thioctic acid derivative having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide, or hydrazide; and each of the other termini of the OEG residue of the conjugatively coupled oligomer composition is stabilizingly and covalently coupled to a therapeutic agent such as a drug, active pharmaceutical agent, polypeptide, protein, enzyme, phospholipid, nucleoside, oligonucleotide, or antibody, said composition having the capability of interacting with a membrane. The thioctic acid portion of the conjugatively coupled oligomer composition may be racemic or may be enriched in one or the other of the two enantiomeric forms of thioctic acid.
In one particular aspect, the present invention relates to a physiologically active therapeutic agent composition comprising a physiologically active therapeutic agent covalently coupled with an oligo(ethylene glycol)-terminated thioctic acid derivative having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide, or hydrazide; and each of the other termini of the OEG residue of the conjugatively coupled oligomer composition is stabilizingly and covalently coupled to a therapeutic agent such as a drug, active pharmaceutical agent, polypeptide, protein, enzyme, phospholipid, nucleoside, oligonucleotide, or antibody, wherein the oligo(ethylene glycol)-terminated thioctic acid derivative moiety and the physiologically active therapeutic agent are conformationally arranged in relation to one another such that the physiologically active therapeutic agent in the physiologically active therapeutic agent composition has an enhanced in vivo resistance to enzymatic modification or degradation, relative to the physiologically active therapeutic agent alone (i.e., in an unconjugated form devoid of the oligo(ethylene glycol)-terminated thioctic acid derivative moiety coupled thereto).
The invention relates in a further aspect to a stable, conjugated therapeutic agent composition comprising a physiologically active therapeutic agent covalently coupled to a physiologically compatible oligo(ethylene glycol)-modified 1,2-dithiolane moiety. In such composition, the physiologically active therapeutic agent may be covalently coupled to the physiologically compatible oligo(ethylene glycol)-modified 1,2-dithiolane moiety by a labile covalent bond, wherein the labile covalent bond is scissionable in vivo by biochemical hydrolysis and/or proteolysis. The physiologically compatible oligo(ethylene glycol)-modified 1,2-dithiolane moiety may advantageously comprise a physiologically compatible oligo(ethylene glycol)-modified lipoic acid ester or amide.
In the above complex, the physiologically active therapeutic agent may, by way of illustration, comprise a peptide, protein, nucleoside, nucleotide, antineoplastic agent, anti-viral agent, anti-resorptive agent, anti-osteoporotic agent, or prodrugs, precursors, intermediates, analogues, or derivatives thereof.
For example, the therapeutic peptide may comprise a peptide selected from the group consisting of insulin, calcitonin, interferons, enkephalins, endorphins, vasopressin, non-naturally occurring opioids, superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, or erythropoietin. The peptide may be human, recombinant, or animal in origin and is obtained and purified by known techniques.
As other examples, the therapeutic agent may comprise an antiviral compound; a cancer chemotherapeutic agent; an antidepressant; an ulcer medication; a cholesterol reducing agent; an opioid such as morphine; or an anti-osteoporotic such as raloxifene or alendronate.
The term Apeptide@ as used herein is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to 10,000. As used herein, the term Acovalently coupled@ means that the specified moieties are either directly covalently bonded to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linage moiety or moieties. The term Aconjugatively coupled@ means that the specified moieties are covalently coupled to one another. The term Atherapeutic agent@ means an agent which is therapeutically useful, e.g., an agent for the prevention, treatment, remission or attenuation of a disease state, physiological condition, symptoms, or etiological factors, or for the evaluation or diagnosis thereof.
The invention thus comprehends various compositions for therapeutic (in vivo) application, wherein the therapeutic agent of the therapeutic agent composition is a physiologically active, or bioactive, therapeutic agent. In such therapeutic agent-containing compositions, the conjugation of the therapeutic agent component to the oligo(ethylene glycol)-terminated dithiolane component may be direct covalent bonding or indirect (through appropriate spacer groups) bonding. Thus, a wide variety of therapeutic agent species may be accommodated in the broad practice of the present invention, as necessary or desirable in a given end use therapeutic application.
In another aspect, therapeutic agent compositions such as those described above may utilize therapeutic agent components intended for diagnostic or in vitro applications, wherein the therapeutic agent is, for example, a diagnostic reagent or a complement of a diagnostic conjugate for immunoassay or other diagnostic or non-in vivo applications. In such non-therapeutic applications, the compositions of the invention are highly usefully employed as stabilized compositions which may, for example, be formulated in compatible solvents or other solution-based compositions to provide stable compositional forms which are of enhanced resistance to degradation.
Also provided in accordance with the invention is a self-assembled monolayer (SAM) composition comprising an activated or reactive, OEG-modified-1,2-dithiolane composition or a conjugate of an OEG-modified-1,2-dithiolane composition adherent to gold, silver, copper, mercury, or an amalgam of these metals. A SAM composition comprising an activated or reactive, OEG-modified-1,2-dithiolane composition or a conjugate of an OEG-modified-1,2-dithiolane composition adherent to gold is most preferred. Further provided are methods for the preparation of these self-assembled monolayers and methods for their dissociation.
The unexpected utility of an activated or reactive, oligo(ethylene glycol)-terminated 1,2-dithiolane composition of the present invention or a conjugate of a reactive, OEG-terminated 1,2-dithiolane composition of the present invention as compared to the utility of the linear OEG-terminated, linear alkyl monothiols known in the art is believed to come from five sources. First, the 1,2-dithiolane segment of a 1,2-dithiolane composition of the present invention reacts with gold or another metal of the present invention to provide a self-assembled monolayer (SAM) composition that is stabilized by multiple sulfur-metal bonds. The multiple sulfur-metal bonds render the resulting SAM composition more stable than that of a monothiol. Second, the other segment of a 1,2-dithiolane composition of the present invention presents at least one activated or reactive terminus available for binding a biological or non-biological receptor, ligand, sequestering, or reporter moiety, or presents at least one terminus to which a biological or non-biological receptor, ligand, sequestering, or reporter moiety may be bound covalently. Third, when bound to the metal surface, a 1,2-dithiolane composition of the present invention is chemically stable in a wide variety of hostile media and conditions. This stability enables presentation of at least one biological or non-biological receptor, ligand or reporter moiety and capture and/or extraction and/or sequestering of a species of interest from a complex environment without undesirable dissociation of the oligo(ethylene glycol)-terminated dithiolane-metal complex during exposure to the hostile environment. Fourth, each of the opposing termini at the end of the OEG-portion of a 1,2-dithiolane composition of the present invention is reactive with, or may be activated to be reactive with, any one of a broad spectrum of electrophilic or nucleophilic reagents. This reactivity enables covalent attachment of a biological or non-biological receptor, ligand, sequestering, or reporter moiety to an activated or reactive, oligo(ethylene glycol)-terminated 1,2-dithiolane composition of the present invention either prior to its attachment to a metal or following its attachment to a metal. Further, if the OEG-portion of a 1,2-dithiolane composition of the present invention is branched, each activated or reactive terminus of an OEG-branch may be joined covalently to a biological or non-biological receptor, ligand or reporter moiety, thereby enabling presentation of a plurality of ligand or reporter moieties. Presentation of a plurality of a biological or non-biological receptor, ligand or reporter moieties is believed to enable more effective binding of a species of interest and its sequestration from a complex environment. Fifth, each composition of the present invention presents a moderately hydrophilic surface (i.e., the OEG-portion of a composition of the present invention) to the external environment. Monolayers of poly- or oligo(ethylene glycol) derivatives are known to minimize non-specific binding of biomolecules to the interactive terminus of the SAM. (C. Pale-Grosdemange, E. S. Simon, K. L. Prime, and G. M. Whitesides. Formation of self-assembled monolayers by chemisorption of derivatives of oligo(ethylene glycol) of structure HS(CH2)11(OCH2CH2)mOH on gold. J. Am. Chem. Soc. 1991; 113: 12-20.)
In addition to the five utilities cited above, a sixth utility has not been heretofore recognized by skilled artisans and applies particularly to the 1,2-dithiolane compositions of the present invention. Application of electrical voltage to a gold-sulfur-terminated reagent complex is known to effect the severance of the gold-sulfur reagent bond and release the reagent as a thiol. With respect to an OEG-terminated 1,2-dithiolane composition of the present invention, application of voltage to a gold-complex of a 1,2-dithiolane composition of the present invention severs both gold-sulfur bonds and releases the composition as the dithiol. Surprisingly, the inventor has found that this dithiol rapidly oxidizes to a ring-closed disulfide (i.e., a 1,2-dithiolane of the present invention).
This unexpected and rapid ring closure to a 1,2-dithiolane composition of the present invention offers distinct advantages to users of the present invention. One significant advantage relates to the relative nucleophilicity and reactivity of thiols compared to the nucleophilicity and reactivity of disulfides. Thiols are nucleophiles, and can undergo a variety of reactions, including, for example, the displacement of another thiol that is part of a disulfide. Thus, release of a thiol enables undesirable displacement reactions to occur, reactions that destroy (i.e., xe2x80x9cscramblexe2x80x9d) existing disulfide bonds that may be critical to the structure and activity of a protein and cause its inactivation or denaturation. (Insulin is an example of a protein in which maintenance of the native disulfide bonds is critical. If insulin is exposed to a thiol, xe2x80x9cscramblingxe2x80x9d of the internal disulfide bonds takes place, and the protein is inactivated.) In contrast, after release from a SAM composition of the present invention, a 1,2-dithiolane of the present invention is re-formed. The disulfide (i.e., 1,2-dithiolane) thus formed is not a nucleophile and does not cause displacement reactions. The lack of chemical reactivity of the 1,2-dithiolane segment of a 1,2-dithiolane of the present invention is advantageous to the user of the present invention in a number of ways, including, by way of example, enabling monitoring of a 1,2-dithiolane composition of the present invention by surface plasmon resonance or mass spectrometry.
A seventh advantage of the 1,2-dithiolanes of the present invention relates specifically to the embodiments in which the 1,2-dithiolane is thioctic acid, d-thioctic acid or a derivative thereof, d-Thioctic acid is a natural substance found in mammals and is an important biological anti-oxidant and enzyme co-factor. Since some of the 1,2-dithiolanes of the present invention are derivatives of d-thioctic acid, it is reasonable to anticipate that these dithiolanes will be physiologically compatible. This is advantageous to the user of the present invention in a number of ways, including, by way of example, enabling use of such a 1,2-dithiolane of the present invention as a means for drug delivery.
The oral route of administration of peptides and proteins is among the most problematic of delivery regimens. Drug delivery via the gastrointestinal (GI) tract requires relatively lengthy exposure to a multi-faceted system that is designed to degrade nutrients and dietary materials into small molecules that are readily transferred from the GI tract into the systemic circulation and to prevent the indiscriminate passage of macromolecules, as well as other large entities such as microbes that may present dangers to the host.
Designing and formulating a polypeptide drug for delivery through the GI tract requires a multitude of strategies. The dosage form must initially stabilize the drug while making it easy to take orally. It must then protect the polypeptide from the extreme acidity and action of pepsin in the stomach. When the drug reaches the intestine, the formulation must incorporate some means for limiting drug degradation by the plethora of enzymes that are present in the intestinal lumen. In addition, the polypeptide and/or its formulations must facilitate both aqueous solubility at near neutral pH and lipid layer penetration in order for the protein to traverse the intestinal membrane and then the basal membrane for entry into the bloodstream. To accomplish this, formulation excipients that promote absorption may be required. Finally, when the modified polypeptide enters the systemic circulation, the structural modifications may add to the functionality of the drug, e.g., by extending its half-life in the circulation. However, any structural changes that may have been employed to enhance oral bioavailability must not interfere with receptor binding and uptake at the site of biological activity.
Therefore, a physiologically active therapeutic agent composition comprising a physiologically active therapeutic agent covalently coupled to a physiologically compatible oligo(ethylene glycol)-modified 1,2-dithiolane moiety wherein the physiologically active therapeutic agent is a peptide or protein and the composition has the ability to interact with biological membranes is a particularly advantageous embodiment of the present invention.
Other aspects, features, and modifications of the invention will be more fully apparent from the ensuing disclosure and appended claims.